spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 8 October 2003
doi: 10.1242/dev.00815

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Müller, M.
Right arrow Articles by Sander, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Müller, M.
Right arrow Articles by Sander, M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Nkx6.1 controls migration and axon pathfinding of cranial branchio-motoneurons

Myriam Müller1, Normund Jabs1, Dietrich E. Lork3, Bernd Fritzsch2 and Maike Sander1,*,{dagger}

1 Center for Molecular Neurobiology, Martinistrasse 85, 20251 Hamburg, Germany
2 Department of Biomedical Sciences, Creighton University, Omaha, NE 68178, USA
3 Institute of Neuroanatomy, University of Hamburg, Martinistrasse 52, 20246 Hamburg, Germany



View larger version (103K):

[in a new window]
  		Fig. 1. Nkx6.1 expression in the developing mouse hindbrain. In situ hybridization with a Nkx6.1 probe on flat-mounted hindbrains at different embryonic stages (A,B,E,F) and on coronal hindbrain sections at E12.5 (G-L). Rhombomeric positions are indicated (r2 to r6). At E8.5 (A), Nkx6.1 is expressed in ventral progenitor cells. At E10.5 (B), Nkx6.1 expression spans the entire length of the ventral hindbrain. At E10.5, immunofluorescence detection of Nkx6.1 (red in C,D) with Nkx2.2 (green in C) or Isl1 (green in D) shows co-expression of Nkx6.1 with both Nkx2.2 and Isl1 (yellow appearing cells in C,D). At E11.5 (E), Nkx6.1 expression is detected in the trigeminal (nV), and in migrating facial branchio-motor (fbm) neurons (nVII) from r4 to r6, as well as in a lateral column representing the motor nuclei of the inner ear efferents (IEE), the superior (nSS) and inferior (nIS) salivatory nucleus and nucleus ambiguous (nA). At E12.5 (F), Nkx6.1 marks the entire stream of migrating fbm neurons from r4 into the dorsal half of r6, and is also detected in the oculomotor (nIII) (G), trochlear (nIV) (H), trigeminal (nV) (I), abducens (nVI) (J), facial nuclei (nVII) (K,J), the nucleus ambiguous (nA) (L), the dorsal motor nucleus of the vagal nerve (dmnX) (L) and the hypoglossal nucleus (nXII) (L). Nkx6.1 is also detected in the raphe nuclei (nRP) (K,L).

 


View larger version (99K):

[in a new window]
  		Fig. 2. Absence of somatic motor nuclei and reduced sizes of branchiomotor nuclei in Nkx6.1 mutant embryos at E18.5. In situ hybridization with a peripherin probe on coronal sections through the hindbrain of wild type (left column) and Nkx6.1 mutant (right column) embryos. In Nkx6.1 mutant embryos, the somatic motor nuclei of the abducens (nVI) (arrow in A,B) and hypoglossal (nXII) (arrow in C,D) nerve are absent. The branchiomotor nuclei of the trigeminal (nV) (E,F) and facial (nVII) (G,H) nerve and the nucleus ambiguous (nA) (I,J) are present in Nkx6.1 mutants, but reduced in size. By contrast, the dorsal motor nucleus of the vagal nerve (dmnX) (arrowhead in C,D) appears normal in size.

 


View larger version (92K):

[in a new window]
  		Fig. 3. Defects in branchio-motoneuron migration and axonal pathfinding in the hindbrain of Nkx6.1 mutant embryos. Application of DiI to the facial (A,B,E,F,G,H) and trigeminal nerve (C,D) in wild-type (A,C,G) and Nkx6.1 mutant embryos (B,D,E,F,H) at E12.5. While facial branchiomotor (bm) neurons are backlabeled along their entire migratory stream from r4 into dorsal r6 in wild-type embryos (A), most facial bm neurons are clustered close to the ventral midline in r4 and few in the rostral third of r5 in Nkx6.1 mutants (B). No difference in the location of facial visceromotor (vm) neurons is observed between wild-type and Nkx6.1 mutant embryos (A,B). In wild-type embryos, trigeminal motoneurons are found at the trigeminal nerve exit point (C), but scattered between the floor plate and the exit point in Nkx6.1 mutants (D). Note the backlabeling of an additional nerve process in Nkx6.1 mutants (D). DiI application to the facial nerve labeled neurons in r2/r3 (E) and r6/r7 (arrowheads in B,F) in Nkx6.1 mutants, but not in wild-type embryos. Simultaneous application of DiI (red) to the facial and DiA (green) to the glossopharyngeal/vagal nerve shows that neurons, which project through the facial nerve in Nkx6.1 mutants (arrowheads in H), are localized within the territory of the inferior salivatory nucleus (nIS) and nucleus ambiguous (nA). (I,J) Lateral view of E10.5 embryos stained with 2H3 anti-neurofilament antibody. The branching pattern of the cranial nerves does not differ between wild-type (I) and Nkx6.1 mutant embryos (J). (K) Schematic summary of the differences in the position and axonal projections of hindbrain motoneurons between wild-type and Nkx6.1 mutant embryos. Trigeminal (V), facial (VII), vestibuloacoustic (VIII), glossopharyngeal (IX), vagal (X), spinal accessory (XI) and hypoglossal (XII) nerves; branchial arch (BA); otic vesicle (OV).

 


View larger version (123K):

[in a new window]
  		Fig. 6. Ectopic expression of cell-surface receptors in facial branchio-motoneurons of Nkx6.1 mutants. Whole-mount in situ hybridization with the indicated probes on flat-mounted hindbrains from wild-type (left columns) and Nkx6.1 mutant (right columns, except J) embryos at the indicated ages. Phox2b marks facial branchio-motor (fbm) neurons (nVII, A-D). At E10.75, few fbm neurons have migrated into r5 in wild type (arrowhead in A) and Nkx6.1 mutants (arrowhead in B). At E12.5, some fbm neurons have completed migration into r6 in wild-type embryos (C), but no neurons are found caudal to upper r5 in Nkx6.1 mutants (D). Tag1 is normally expressed in fbm neurons in r4 in both wild-type (E) and Nkx6.1 mutant embryos (F). Ret, which is normally restricted to migrating fbm neurons in r5 and r6 (G), is ectopically expressed in r4 in Nkx6.1 mutants (H). In wild-type embryos, Unc5h3 is first detected at E12.0 (I) in facial and trigeminal (nV) motoneurons, and motoneurons of the superior (nSS) and inferior salivatory nucleus (nIS), as well as the nucleus ambiguous (nA). The expression of Unc5h3 in these neurons is maintained at E13.5 (J). Neogenin is expressed in dorsolaterally migrating facial and trigeminal motoneurons (O). In Nkx6.1 mutants, fbm neurons ectopically express Unc5h3 (L) and do not express neogenin (P). In situ hybridization for Unc5h3 on coronal sections through r4 verifies that the ectopic expression is specific to fbm neurons (N). Abducens nucleus, nVI.

 


View larger version (126K):

[in a new window]
  		Fig. 4. Rhombomere identity is not affected by the absence of Nkx6.1 activity. In situ hybridization on flat-mounted hindbrains at E10.5. The expression pattern of Hoxb1 (A,B), and Epha4 (C,D) is similar in wild-type (A,C) and Nkx6.1 mutant embryos (B,D).

 


View larger version (105K):

[in a new window]
  		Fig. 5. Specification of facial branchio-motoneurons is not affected by Nkx6.1 inactivation. In situ hybridization with Isl1 (A,B) and Isl2 (D,E) on flat-mounted hindbrains of E10.5 wild-type (A,D) and Nkx6.1 mutant (B,E) embryos. (B) Nkx6.1 mutant embryos generate Isl1-positive cells throughout the hindbrain. (C) Quantification of Isl1-positive facial branchio-motoneurons at r4 levels of wild-type and Nkx6.1 mutant embryos at E10.5 and E12.5. Using immunohistochemistry with an anti-Isl1 antibody on coronal hindbrain sections, Isl1-positive nuclei on 12 representative sections for each genotype and age were counted. Values are shown as % of wild type, mean±s.d. (D,E) Isl2 marks somatic motoneurons and the otic ganglion. Isl2-positive motoneurons are not detected in hindbrains of Nkx6.1 mutants (E). (F-S) In situ hybridization with Olig2 (F,G), Irx3 (H,I), Dbx2 (J,K), Dbx1 (L,M) and Ebf1 (R,S) and co-immunofluorescence detection of En1 (N,O) or Evx1 (P,Q) together with Phox2b (N-Q) on sections through r4 of wild-type (F,H,J,L,N,P,R) and Nkx6.1 mutant embryos (G,I,K,M,O,Q,S) at E10.5. These markers are similarly expressed in wild-type and in Nkx6.1 mutant embryos.

 


View larger version (69K):

[in a new window]
  		Fig. 7. Facial branchio-motoneurons in r4 co-express Nkx6.1 and Nkx6.2. Immunofluorescence detection of Nkx6.2 (red in A,B and green in C,D) with Nkx2.2 (green in A), Isl1 (green in B), Nkx6.1 (red in C,D) on sections through r4 in wild-type (A-C) and Nkx6.1 mutant (D) embryos at E10.5. Nkx6.2 is co-expressed with Nkx2.2 (yellow appearing cells in A), Isl1 (yellow appearing cells in B) and Nkx6.1 (yellow appearing cells in C). The number of Nkx6.2-expressing cells and the level of Nkx6.2 expression are not changed in Nkx6.1 mutants (D). (E-H) Whole-mount in situ hybridization with Nkx6.2 on flat-mounted hindbrains from E11.0 (E,F) and E12.5 (G,H) wild-type (E,G) and Nkx6.1 mutant embryos (F,H). At E11.0, Nkx6.2 is strongly expressed in facial branchio-motor (fbm, nVII) neurons in r4, but not in migrating fbm neurons in r5 or r6 (E). At E12.5, expression of Nkx6.2 in fbm neurons is markedly downregulated (G). Trigeminal branchio-motoneurons (nV) express Nkx6.2 at E11.0 (E) and E12.5 (G). Nkx6.2 expression in fbm neurons does not differ between wild-type and Nkx6.1 mutant embryos (E-H). However, in Nkx6.1 mutants, Nkx6.2 does not mark the trigeminal branchio-motoneurons at E11.0 (F), and is only faintly detected in these neurons at E12.5 (H). The signal may be decreased in Nkx6.1 mutants, because trigeminal neurons are not clustered as a compactly as in wild-type embryos.

 


View larger version (31K):

[in a new window]
  		Fig. 8. Summary of the molecular changes in facial branchio-motoneurons of Nkx6.1 mutant embryos. (A) Between E10.5 and E11 pre-migratory facial branchio-motor (fbm) neurons in r4 express both Nkx6.1 and Nkx6.2. Early migratory populations of fbm neurons progress into rostral r5 in both wild-type and Nkx6.1 mutant embryos, and do not express the cell-surface receptors Unc5h3, Ret and neogenin. (B) Although Nkx6.2 is strongly expressed at the early stages of fbm neuron migration, later populations of pre-migratory fbm neurons at E12.5 express only low levels of Nkx6.2. At E12.5, some fbm neurons have already migrated from r4 into r6, and Unc5h3, Ret and neogenin are expressed in these neurons in a rhombomere-specific pattern. In Nkx6.1 mutants, fbm neurons do not migrate caudally, and show ectopic expression of Unc5h3 and Ret in r4.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2003